To understand the interleukin 1 converting enzyme (ICE), it is helpful to first examine the role of interleukin-1 (IL-1), its enzymatic substrate. IL-1 facilitates host natural immunity, predominantly those aspects related to the initiation of inflammatory reactions that protect the body against bacterial infection. (Ayala et al. (1994) J Immunol 53: 2592-2599). At low concentrations in the bloodstream, IL-1 mediates local inflammation by inducing the synthesis of other cytokines, such as IL-6 and IL-8, and the synthesis of proteins that mediate leukocyte adhesion, and prostaglandin production. (Abbas et al. (1994) Cellular and Molecular Immunology, W B Saunders Company). At intermediate concentrations in the bloodstream, IL-1 may induce fever, the synthesis of acute plasma proteins by the liver, and metabolic wasting, cachexia (Abbas, supra). At even higher concentrations, IL-1 has been implicated in tissue destruction observed in numerous inflammation-related diseases, including rheumatoid arthritis, septic shock, inflammatory bowel disease and insulin-dependent diabetes mellitus. (Li et al. (1995) Cell 80:401-411).
IL-1 activity results from the expression and release of two gene products, IL-1.alpha. and IL-1.beta., predominantly from activated monocytes. (Howard et al. (1991) J Immunology 147:2964-2969). Both gene products are initially synthesized as inactive precursors of about 31 kD in monocytes. Pre-IL-1.beta. is cleaved to an active 17 kD form by the IL-1.beta.-converting enzyme (ICE) before release from activated monocytes. On the other hand, pre-IL-1.alpha. is likely cleaved to an active 17 kD form by a calpain-like, IL-1.alpha.-converting enzyme prior to release. (Carruth et al. (1991) J Biol Chem 266:12162-12167). Additionally, ICE has been implicated in the release of IL-1.alpha. from activated monocytes but the mechanism is not understood (Li, supra).
The IL-1.beta. gene product is the predominant form of IL-1 that is present at high concentrations in the bloodstream during inflammatory diseases, such as rheumatoid arthritis, septic shock, inflammatory bowel disease, and insulin-dependent diabetes mellitus (Li, supra). Since the cleavage of pre-IL-1.beta. by ICE is coupled to IL-1.beta. release and to increased IL-1 activity in the bloodstream, ICE activity may be higher in these pathological conditions.
The importance of regulating ICE activity to modulate the IL-1.beta. concentration to affect the host immune response has recently been confirmed: the crmA gene product of cowpox virus prevents the proteolytic activation of IL-1.beta. and inhibits the host inflammatory response. Cowpox virus containing a deleted crmA gene is unable to suppress the inflammatory response, resulting in a reduction of virus-infected cells and less damage to the host. (Miura et al. (1993) Cell 75: 653-660).
ICE is a novel cysteine protease that is known specifically to cleave inactive IL-1.beta. precursor to its active form. (Ayala, supra). This protease recognizes the sequence Asp-X, where X is preferably a small hydrophobic amino acid residue, and cleaves the bond between Asp and X. However, many Asp-X bonds are not recognized by ICE suggesting that flanking sequences are also required for recognition and cleavage. In the case of IL-1.beta., ICE cleaves the precursor to form active IL-1.beta. at two sequence-specific bonds: the bond between residues Asp-27 and Gly-28 and the bond between residues Asp-116 and Ala-117.
ICE itself is synthesized and maintained in cells as an inactive 45 kD precursor which is processed into the active ICE consisting of 20- and 10-kD subunits, p20 and p10. (Ayala, supra). The 45 kD precursor which is cleaved into four different fragments: a 13 kD precursor domain, the p20, a 2 kD spacer, and the p10. Since all these polypeptide fragments are flanked by Asp-X residues in the intact 45 kD precursor, it is possible that the ICE precursor is activated autocatalytically. (Ayala, supra).
The three dimensional structure of ICE has been determined from crystallographic studies. (Walker et al. (1994) Cell 78:343-352). First, it is apparent that the active form of ICE is a homodimer of catalytic domains, each of which consists of p20 and p10 subunits. Second, although the active site cysteine residue is located on p20, both p20 and p10 are essential for activity. p20 and p10 structures are intertwined so as to create a unique 6-stranded, .beta.-sheet core flanked on either side by .alpha. helices. The first 4 .beta. strands ,are contributed by p20, while the remaining 2 .beta. strands are contributed by p10.
The ICE gene from various sources has been sequenced and possesses homology (29% homology overall) to the product of a gene with a possible role in apoptosis: the Caenorhabditis elegans gene ced-3. (Yuan et al. (1993) Cell 75: 641-652). Additionally, the ICE gene contains a sequence region, spanning residues 166 to 287 of the human ICE gene, which shares a 43% homology with ced-3. It is not known whether ced-3 acts as a cysteine protease but it contains the purported catalytic residues that are located at the ICE active site (Cys.sub.285 and His.sub.237). The amino acid pentapeptide Glu--Ala--Cys--Arg--Gly (QACRG), containing the active site cysteine, is the longest oligopeptide conserved among ICE from mice and humans and CED-3 from three different nematodes. Additionally, ced-3 contains the same four residues whose side chains are implicated in binding the aspartate carboxylate group of the substrate at the catalytic site, Arg.sub.179, Gln.sub.283, Ser.sub.347, and Arg.sub.341 (Yuan, supra).
Inhibition with the ICE-specific inhibitor crmA blocks TNF- and FAS-induced apoptosis. Therefore, ICE or a homolog of the molecule is believed to be involved in TNF-and FAS-induced apoptosis.
Additionally, ICE possesses a degree of homology to a gene product with a possible role in embryogenesis: the mammalian gene Nedd-2/lch-1 is expressed during embryonic brain development and is down-regulated in the adult brain. (Yuan, supra). Nedd-2, ced-3, and ICE gene products are about 27% homologous with the carboxyterminus of CED-3 and p10 possessing the highest degree of homology to Nedd-2. The Nedd-2 gene product does not contain the highly conserved QACRG pentapeptide so Nedd-2 probably is not a cysteine protease.
To confirm ICE's role in inflammation-related diseases by controlling the levels of active IL-1.beta., ICE-deficient knockout mice were created (Li, supra). These genetically-engineered mice were normal physiologically but lacked the ability to process precursor IL-1.beta. to its active form when monocytes were activated with microbial products, such as lipopolysaccharide (LPS). Additionally, the production of IL-1.alpha. was decreased, and the level of other cytokines, tumor necrosis factor (TNF) and IL-6, involved in inflammatory responses to microbial products was somewhat reduced. These mice were resistant to the lethal effects of septic shock when exposed to LPS (Li, supra). Therefore, inhibiting ICE activity to lower the concentration of IL-1.beta. in the bloodstream may be a method of treating inflammation-related diseases. ICE also may help identify patients who are susceptible to these diseases.
Since ICE shares sequence homology to ced-3 and overexpression of ICE appears to induce apoptosis, the ICE-deficient mice studies were important because the mice seemed normal in terms of their development. If ICE itself played a strong role in apoptosis during development, the ICE-deficient mice should have had gross abnormalities in brain, gut, lymphoid and brain tissues, and have autoimmune diseases (Li, supra). This implies that other ICE-like proteins are involved in these aspects. However, ICE may perform functions other than IL-1.beta. precursor cleavage. ICE mRNA has been detected in a greater variety of tissues than IL-1.beta. mRNA has (Miura, supra).
ICE has attracted interest as a target for novel anti-inflammatory drugs, because the cytokine which it activates, IL-1.beta., is proinflammatory and has been implicated in the pathophysiology of various diseases, including rheumatoid arthritis, septic shock, inflammatory bowel disease and insulin-dependent diabetes mellitus (Dinarello and Wolff (1993) N Engl J Med 328:106-13). The provision of a new ICE gene and polypeptide will further drug research in screening for and designing more effective and more specific inhibitors to this pro-inflammatory substance. The ICE molecule which is the subject of this patent application was identified among the sequences of a cDNA library made from human lung. A short description of the organ and its cells follows.
The Lung
The respiratory system is composed of 1) a ventilation mechanism, 2) a conduction passageway comprising the nasal cavity, nasopharynx, larynx, trachea, bronchi, bronchioles, and terminal bronchioles which cleans, moistens, and warms incoming air, and 3) a respiratory portion of the lungs comprising the respiratory bronchioles, alveolar ducts, and alveoli where gas exchange takes place.
Respiratory Mucosa
Beginning in the nasal cavity and extending through the larger bronchioles, air passageways are coated with a viscoelastic sheet produced by mucous and serous cells located in the surface epithelium and underlying submucosal glands. The surface epithelium is pseudostratified and typically contains mucus-producing goblet cells, ciliated columnar cells, brush cells, basal cells, and small granule cells.
Mucins are large glycoproteins which form the structural components of the viscoelastic layer. They are synthesized in intracellular granules in surface goblet cells and the mucous cells of the submucosa glands. The release of mucin is accompanied by swelling and gel formation similar to that occurring in the intestinal tract. Water and ions primarily from ciliated cells accomplish the hydration.
Serous cells, which comprise the majority of cells in the submucosal glands, make and secrete immunoglobulin A (IgA). Dense granules in the serous cells contain lysozyme and antiproteases, which inactivate destructive bacterial enzymes. The antiproteases also destroy proteases released by neutrophils before they can cause the secondary damage common to pulmonary disease. Within serous cell granules the positively charged secretory proteins are bound to negatively charged sulfated proteoglycans. Opposite-charge packing, common to many granules, is a mechanism that concentrates contents by reducing osmotic activity to exclude water. In addition to their structural role within the granule, proteoglycans released with secretory proteins during exocytosis are incorporated into the mucin layer and may make important contributions to its physical substructure.
The blanket-like secretion covering the epithelium is continually moved towards the pharynx by the coordinated activity of the cilia which project from up to 90% of the surface epithelial cells. These cilia move in an undulating wave in a low-viscosity layer under the viscoelastic sheet. Organisms and particles trapped within the mucous layer are carried to the pharynx, where they are swallowed or expectorated. This "clearance" of the airway is the primary factor preventing respiratory damage and infection. In respiratory diseases such as asthma, chronic bronchitis, and cystic fibrosis there is a hypersecretion of mucus and hyperplasia of goblet cells. Excess mucus obstructs the airway, halting the clearing feature; if bacteria and viruses accumulate and overwhelm cellular defense, infection results.
Bronchioles are located between the larger airways which contain cartilage and extensive submucosal glands and the delicate alveoli where gas exchange occurs. The alveoli have a high proportion of circularly arranged smooth muscle and are lined with epithelial cells. In the terminal bronchioles, the epithelium is cuboidal and consists of ciliated and nonciliated cells. The ciliated cells move secretions and trapped airborne particles towards the pharynx. The nonciliated cells are unique to bronchioles and their major function is secretion of the material lining the bronchiolar lumen. This material contains proteins important in defense (eg, lysozyme and antibodies) and in breaking up the mucus produced in the upper airway. Typically, nonciliated cells have an apical region of densely packed granules and large mitochondria and a basal portion which contains the nucleus, rough endoplasmic reticulum (ER), and patches of glycogen. The cells' smooth ER may function to detoxify a variety of compounds, and the granules may have lysosomal function for recycling secretions.
Of all the respiratory passageways, the bronchiole is occluded most easily. Because of the small size of the bronchiolar lumen, factors associated with disease, such as spasmodic contraction of smooth muscles (as in asthma) or abnormal production of mucus (as in chronic bronchitis due to smoking) can close bronchioles and reduce airflow enough to be life threatening. During an allergic response, parasympathetic nerves that innervate the smooth muscle of the bronchioles release acetylcholine, a bronchoconstrictor. Bronchioles are also particularly sensitive to mediators, such as leukotrienes from mast cells.
Gas exchange occurs in the over 300 million bubble-like alveoli at the end of the respiratory passageways. Type I alveolar cells, which are simple squamous cells, and Type II alveolar cells, which are intermittent cuboidal secretory cells, line the alveoli and are continuous with the cuboidal epithelium lining the terminal bronchioles. A network of pulmonary capillaries surround each alveolus. Oxygen and carbon dioxide diffuse across the cell layers of the alveoli which form the blood-air barrier into the capillary blood and carbon dioxide diffuses in the opposite direction. Carbonic anhydrase present in red blood cells in the capillaries liberates CO.sub.2 from H.sub.2 CO.sub.3.
The interalveolar septum is composed of five main cell types: capillary endothelial cells (30%); Type I alveolar cells (8%); Type II alveolar cells (16%); interstitial cells, including fibroblasts and mast cells (36%); and alveolar macrophages (10%). Macrophages are present within the air space and in the interstitial tissue of the alveolar septa. When activated by airborne irritants, macrophages recruit blood cells such as lymphocytes to aid in their defensive efforts. Eventually, as the disease state progresses, blood cells can enter the air space to join the macrophages, resulting in congestive heart failure.
The primary role of the interstitial fibroblast of the lung is the maintenance of the integrity of the alveolar compartment by its production of collagens, predominantly Types I and III collagen, elastin fibers, and other matrix components, including fibronectin and proteoglycans (PGs). Human lung fibroblasts secrete the two small chondroitin/dermatan sulfate PGs, PG-I (biglycan, 300 kD) and, in larger proportion, PG-II (decorin, 130 kD). Transforming growth factor-beta (TGF-.beta.), which acts as a growth inhibitor, selectively induces the expression of PG-I, but not PG-II. PG-I and PG-II may act as mediators of the growth inhibition caused by TGF-.beta. (Romaris et al (1991) Biochim Biophys Acta 1093:229-33).
Collagen supports the septum, and elastin fibers accommodate the stretching associated with the respiration. Fibroblasts also synthesize a variety of enzymes, including collagenase, and products that may modulate the function of other cells. These include chemotactic factors, prostaglandins, tissue plasminogen activator (tPA), components of the complement system, and superoxide dismutase. During early lung development and under the regulation of glucocorticoids, fibroblasts produce a pneumocyte factor that stimulates the synthesis of surfactant by alveolar type 2 cells. In the event of injury to the septum, fibroblasts divide and secrete more collagen for repair. Chronic insult leads to the overproduction of collagen and to scarring which interferes with gas exchange.
Attenuated Type I alveolar cells are particularly susceptible to damage but are not capable of replacing themselves following injury. Repair and routine replacement is carried out by Type II alveolar cells which suspend their secretory activity, divide and differentiate into Type I cells. With chronic injury, Type II cells divide but do not differentiate into Type I cells. As a result, parts of the air spaces become lined by cuboidal Type II cells which reduces the area available for gas exchange.
Type II alveolar cells are responsible for the secretion and turnover of surfactant, a macroaggregate of phospholipids (primarily dipalmitoylphosphatidylcholine) and proteins, which coats the entire alveolar surface. The surfactant reduces the surface tension of water molecules covering the alveolar surface, prevents the collapse of the lung during exhalation, and reduces the amount of energy necessary to reinflate the lung. Growth factors, hormones and simple mechanical stretching stimulate secretion of surfactant. With each inhalation the alveolar surface area may increase as much as 80%, and the secretion of surfactant must cover the expanded area. In order to maintain a thin, surface-active film during exhalation, the excess surfactant is removed by the endocytotic pathways of Type II cells.
About 10% of surfactant is protein, including a family of large, acidic glycoproteins (SP-A) essential to the recycling of surfactant and two smaller hydrophobic peptides (SP-B, SP-C) that promote surfactant spreading. At birth, the maturity of the lungs and adequate surfactant are critical to prevent collapse at first breath. Hyaline membrane disease and respiratory distress syndrome involve inadequate surfactant which may be overcome by administration of glucocorticoids at critical stages of development and/or surfactant replacement at birth.
Fluids do not normally enter the air space because of negative osmotic pressure in the interstitium and diversion of excess fluid into the lymph system. However, if endothelial cells are damaged and excess protein leaks into the interstitium, osmotic pressure changes from negative to positive, the Type I cell layer is damaged, and fluids leak into the alveoli.
An extensive treatment of lung cell biology may be found in Massaro (1989, Lung Cell Biology, Marcel Dekker Inc, New York N.Y.), incorporated herein by reference.
Disorders of the Lung
Emphysema is a disorder of the lung characterized by destruction of the walls of the alveoli. Consequent enlargement of the distal airspaces leads to impaired ability of the cardiopulmonary system to deliver oxygen to other organs. Individuals suffering with emphysema typically have difficulty exhaling due to the loss of elastic recoil and airway support normally provided by the alveoli. Moreover, the loss of alveolar tissue diminishes the surface area available for gas exchange and the loss of pulmonary capillaries limits the capacity of the right heart to transfer the cardiac output across the lungs.
Emphysema can result from a common lethal hereditary disorder, .alpha.1-antitrypsin (.alpha.1-AT) deficiency. Neutrophil elastase which is released by activated or lysed neutrophils, cleaves the major connective tissue components of the alveolar walls, including elastin, collagen types I, III, and IV, laminin, fibronectin, and the protein components of proteoglycans. In normal individuals, neutrophil elastase is inhibited by .alpha.1-AT and by secreted leukoprotease inhibitor, a 12 kD protein produced by airway secretory cells. Uninhibited neutrophil elastase also interacts with receptors on the surface of alveolar macrophages which results in their activation. Activated macrophages release leukotriene B4, a potent neutrophil chemoattractant, further increasing the burden of neutrophils and of neutrophil elastase in the lung. The presence of uninhibited neutrophil elastase in the lower respiratory tract causes progressive destruction of the alveolar walls.
Emphysema is common among cigarette smokers. Cigarette smoke induces alveolar macrophages to release chemoattractants for neutrophils and causes the release of free radicals, such as superoxide and hydrogen peroxide, which oxidize the Met 358 residue of .alpha.1-AT, inactivates .alpha.1-AT and results in alveolar damage.
Bronchitis
Bronchitis is a disorder characterized by inflammation of the airway epithelium, generally involving both large and small airways. The inflammation causes mucous hypersecretion, leading to dysfunction of host defenses and bacterial colonization of the retained secretions and infections. In some individuals, the inflammation is sufficiently extensive that it is characterized by progressive derangement of the airway architecture, loss of lung function, and respiratory limitation.
The most aggressive form of bronchitis is cystic fibrosis (CF), which is caused by a mutation of the CF transmembrane regulator (CFTR). Affected individuals develop frequent respiratory infections localized to the epithelial surface of the airways. Eventually those affected by CF develop the chronic production of thick, sticky sputum which, in turn, is colonized with bacteria. Thus CF is characterized by chronic inflammation and infection of the airways, and progressive deterioration of airway function, derangement of pulmonary parenchyma, and finally respiratory failure. In individuals suffering with CF, the inflammation on the airway epithelial surface is so intense that both .alpha.1-AT and leukoprotease inhibitor defenses are overwhelmed and rendered ineffective. Moreover, there are decreased levels of glutathione, an antioxidant, in the fluid covering the respiratory epithelial surface. As a result the epithelium develops chronic, oxidant-induced damage.
The most common form of bronchitis is that associated with cigarette smoking. It has features similar to those of the bronchitis observed in CF, although in a milder form. The inflammatory population includes neutrophils and alveolar macrophages which release an increased burden of oxidants and proteases, including neutrophil elastase, on the airway epithelial surface.
Mechanisms of respiratory tissue injury from cigarette smoking are reviewed in McCusker (1992) Amer J Med 93S:18-21, and inflammatory lung diseases are reviewed in Jennings and Crystal (In: Gallin et al. (1992) Inflammation: Basic Principles and Clinical Correlates, Raven Press, New York N.Y.).
Pulmonary Fibrosis
In pulmonary fibrosis, alveolar walls are thickened by scarring of the interstitium as a result of inflammatory disorders of the lower respiratory tract. Pulmonary fibrosis increases the work required for inspiration and reduces the surface area of the pulmonary capillary bed; transfer of oxygen to the pulmonary capillaries and other organs is limited. Alveolar macrophages, neutrophils, lymphocytes, and eosinophils dominate to variable degrees in different disorders.
The proliferation and function of lung fibroblasts can be directly stimulated or inhibited by a variety of substances that cause lung damage which can eventually lead to interstitial pulmonary fibrosis. These substances include mineral particulates such as silica and asbestos, the metal beryllium, and the antineoplastic drug bleomycin. Idiopathic pulmonary fibrosis (IPF) is a form of pulmonary fibrosis which may result from alveolar inflammation initiated by IgG immune complexes (Jennings and Crystal, supra).
Asbestos exposure, for example, results in lung lesions extending along the alveolar and duct walls that are characterized by a diffuse thickening of the tissue due to increased deposition of connective tissue components. Early lung response to a variety of pneumotoxic agents is characterized by an initial interaction of alveolar and interstitial macrophages with the asbestos fibers leading to complement activation and the production of C5a, a potent chemotactic factor for macrophages and neutrophils, followed by an influx of inflammatory and immune cells, into the exposed tissues.
Asbestosis is characterized by pulmonary cellular hyperplasia, inflammation, and accumulation of connective tissue matrix. Macrophages present at and recruited to the site of asbestosis exposure secrete a number of potent inflammatory mediators and cytokines, including various arachidonic acid metabolites, including prostaglandins and leukotrienes, and bioactive peptides such as platelet-derived growth factor, TGF-.beta., macrophage-derived growth factor, and IL-1. The resulting proliferation of lung fibroblasts eventually leads to fibrosis.
Interstitial pulmonary fibrosis caused by asbestos exposure is reviewed by Brody (In: Gallin et al. (1992) Inflammation: Basic Principles and Clinical Correlates, Raven Press, New York N.Y.).
Asthma
Asthma is characterized by a reversible airways obstruction or inflammation which is attributed to hypersensitivity to stimuli. Symptoms include the wheezing, coughing, and shortness of breath in a mild attack which may progress through three worsening stages all the way to respiratory arrest. Asthma attacks are due to spasm of smooth muscle, edema of the mucosa, increased mucous secretion, eosinophilic infiltration of the mucosa and walls, and injury including desquamation of the epithelium. Both the prevalence and mortality from asthma has increased worldwide.
Typically all asthmatics with active disease have hyper-responsive airways and an exaggerated bronchoconstrictor response to many different stimuli which may include exercise. Mast cells are involved in the response to different inhaled allergens. Eosinophils contribute to the pathogenesis of chronic inflammation by releasing proteins capable of damaging the epithelium. Although the role of neutrophils is unknown, macrophages, lymphocytes and their secretions have been implicated in inflammation. Examples of secreted molecules include histamine, leukotrienes, prostaglandins, platelet activating factor, etc.
Neurogenic/cholinergic factors also influence the bronchorestrictive response. When irritants are inhaled, sensory neurons release substance P, neurokinin A, and calcitonin-related peptide. Etiologic factors, even for allergy-induced asthma, are often difficult to assess, and attacks may be complicated by emotional components such as stress. Nonspecific allergic irritants include cigarette smoke, viral and bacterial infections, pollens, molds, epidermal scales, dust mites, etc.
Drug therapy includes five useful groups--.beta.-adrenergic agents (epinephrine, isoproterenol, terbutaline, albuterol, etc) which relax smooth muscle and inhibit mediator release; theophylline which relaxes smooth muscle and inhibits release of calcium and other chemical mediators; corticosteroids which inhibit leukocytes and leukotriene release; chromolyn sodium which inhibits mediator release and is useful for maintenance therapy; and anticholinergic agents such as atropine which block cholinergic pathways and provide bronchodilation.
These and other respiratory diseases are discussed more fully in The Merck Manual of Diagnosis and Therapy (1992) Merck Research Laboratories, Rahway, N.J. and in Seaton et al. (1989) Crofton and Douglas's Respiratory Diseases, Blackwell Scientific Publications, Boston.